The invention relates to compounds which contain an antigen binding region which is bound to at least one enzyme which is able to metabolize a compound (prodrug) which has little or no cytotoxicity to a cytotoxic compound (drug), where the antigen binding region is composed of a single polypeptide chain. It is advantageous for covalently bonded carbohydrates to be present on the polypeptide chain.
The combination of prodrug and antibody-enzyme conjugates for use as therapeutic composition has already been described in the specialist literature. This entails antibodies which are directed against a particular tissue and to which a prodrug-cleaving enzyme is bound being injected into an organism, and subsequently a prodrug compound which can be activated by the enzyme being administered. The action of the antibody-enzyme conjugate bound to the target tissue is intended to convert the prodrug compound into a compound which exerts a cytotoxic effect on the bound tissue. However, studies on antibody-enzyme conjugates have shown that these chemical conjugates have unfavorable pharmacokinetics so that there is only inadequate site-specific tumor-selective cleavage of the prodrug. Some authors have attempted to remedy this evident deficiency by additional injection of an anti-enzyme antibody which is intended to bring about rapid elimination of the anti-body-enzyme conjugate from the plasma (Sharma et al., Brit. J. Cancer, 61, 659, 1990). Another problem of antibody-enzyme conjugates is the limited possibility of preparing large amounts reproducibly and homogeneously.
The object of the present invention was now to find fusion proteins which can be prepared on an industrial scale and are suitable, by reason of their pharmacokinetic and pharmacodynamic properties, for therapeutic uses.
It has been found in this connection that compounds which contain an antigen binding region which is composed of a single polypeptide chain have unexpected advantages for the preparation and use of fusion proteins, to which carbohydrates are advantageously attached, in prodrug activation.
The invention therefore relates to compounds which contain an antigen binding region which is bound to at least one enzyme, where the antigen binding region is composed of a single polypeptide chain, and carbohydrates are advantageously attached to the fusion protein.
An antigen binding region means for the purpose of the invention a region which contains at least two variable domains of an antibody, preferably one variable domain of a heavy antibody chain and one variable domain of a light antibody chain (sFv fragment). The antigen binding region can, however, also have a bi- or multivalent structure, i.e. two or more binding regions, as described, for example, in EP-A-0 404 097. However, a human or humanized sFv fragment is particularly preferred, especially a humanized sFv fragment.
The antigen binding region preferably binds to a tumor-associated antigen (TAA), with the following TAAs being particularly preferred:                neural cell adhesion molecule (N-CAM),        polymorphic epithelial mucin (PEM),        epidermal growth factor receptor (EGF-R),        Thomsen Friedenreich antigen β (TFβ),        gastrointestinal tract carcinoma antigen (GICA),        ganglioside GD3 (GD3),        ganglioside GD2 (GD2),        Sialyl-Lea, Sialyl-Lex,        TAG72,        the 24-25 kDa glycoprotein defined by MAb L6,        CA 125 and, especially,        carcinoembryonic antigen (CEA).        
Preferred enzymes are those enzymes which are able to metabolize a compound of little or no cytotoxicity to a cytotoxic compound. Examples are β-lactamase, pyroglutamate aminopeptidase, galactosidase or D-aminopeptidase as described, for example, in EP-A2-0 382 411 or EP-A2-0 392 745, an oxidase such as, for example, ethanol oxidase, galactose oxidase, D-amino-acid oxidase or α-glyceryl-phosphate oxidase as described, for example, in WO 91/00108, peroxidase as disclosed, for example, in EP-A2-0 361 908, a phosphatase as described, for example, in EP-A1-0 302 473, a hydroxynitrilelyase or glucosidase as disclosed, for example, in WO 91/11201, a carboxypeptidase such as, for example, carboxypeptidase G2 (WO 88/07378), an amidase such as, for example, penicillin 5-amidase (Kerr, D. E. et al. Cancer Immunol. Immunther. 1990, 31) and a protease, esterase or glycosidase such as the already mentioned galactosidase, glucosidase or a glucuronidase as described, for example, in WO 91/08770.
A β-glucuronidase is preferred, preferably from Kobayasia nipponica or Secale cereale, and more preferably from E. coli or a human β-glucuronidase. The substrates for the individual enzymes are also indicated in the said patents and are intended also to form part of the disclosure content of the present application. Preferred substrates of βglucuronidase are N-(D-glycopyranosyl)benzyloxycarbonylanthracyclines and, in particular, N-(4-hydroxy3-nitrobenzyloxycarbonyl)doxorubicin and daunorubicin β-D-glucuronide (J. C. Florent et al. (1992) Int. Carbohydr. Symp. Paris, A262, 297 or S. Andrianomenjanahary et al. (1992) Int. Carbohydr. Symp. Paris, A 264, 299).
The invention further relates to nucleic acids which code for the compounds according to the invention. Particularly preferred is a nucleic acid, as well as its variants and mutants, which codes for a humanized sFv fragment against CEA (carcinoembryonic antigen) linked to a human β-glucuronidase, preferably with the nucleotide sequence of SEQ ID NO:1, which codes for the amino acid sequence of SEQ ID NO2 (sFv-huβ-Gluc).
The compounds according to the invention are prepared in general by methods of genetic manipulation which are generally known to the skilled worker, it being possible for the antigen binding region to be linked to one or more enzymes either directly or via a linker, preferably a peptide linker. The peptide linker which can be used is, for example, a hinge region of an antibody or a hinge-like amino-acid sequence. In this case, the enzyme is preferably linked with the N terminus to the antigen binding region directly or via a peptide linker. The enzyme or enzymes can, however, also be linked to the antigen binding region chemically as described, for example, in WO 91/00108.
The nucleic acid coding for the amino-acid sequence of the compounds according to the invention is generally cloned in an expression vector, introduced into prokaryotic or eukaryotic host cells such as, for example, BHK, CHO, COS, HeLa, insect, tobacco plant, yeast or E.coli cells and expressed. The compound prepared in this way can subsequently be isolated and used as diagnostic aid or therapeutic agent. Another generally known method for the preparation of the compound according to the invention is the expression of the nucleic acids which code therefor in transgenic mammals with the exception of humans, preferably in a transgenic goat.
BHK cells transfected with the nucleic acids according to the invention express a fusion protein (sFv-huβ-Gluc) which not only was specific for CEA but also had full β-glucuronidase activity (see Example 5).
This fusion protein was purified by anti-idiotype affinity chromatography in accordance with the method described in EP 0 501 215 A2 (Example M). The fusion protein purified in this way gives a molecular weight of 100 kDA in the SDS PAGE under reducing conditions, while molecules of 100 and 200 kDa respectively appear under non-reducing conditions.
Gel chromatography under native conditions (TSK-3000 gel chromatography) showed one protein peak (Example 6, FIG. I) which correlates with the activity peak in the specificity enzyme activity test (EP 0 501 215 A2). The position of the peak by comparison with standard molecular weight markers indicates a molecular weight of ≈200 kDa. This finding, together with the data from the SDS PAGE, suggests that the functional enzymatically active sFv-huβ-Gluc fusion protein is in the form of a “bivalent molecule”, i.e. with 2 binding regions and 2 enzyme molecules. Experiments not described here indicate that the fusion protein may, under certain cultivation conditions, be in the form of a tetramer with 4 binding regions and 4 enzyme molecules. After the sFv-huβ-Gluc fusion protein had been purified and undergone functional characterization in vitro, the pharmacokinetics and the tumor localization of the fusion protein were determined in nude mice provided with human gastric carcinomas. The amounts of functionally active fusion protein were determined in the organs and in the tumor at various times after appropriate workup of the organs (Example 7) and by immunological determination (triple determinant test, Example 8). The results of a representative experiment are compiled in Table 1.
Astonishingly, a tumor/plasma ratio of 5/1 is reached after only 48 hours. At later times, this ratio becomes even more favorable and reaches values >200/1 (day 5). The reason for this favorable pharmacokinetic behavior of the sFv-huβ-Gluc fusion protein is that fusion protein not bound to the tumor is removed from the plasma and the normal tissues by internalization mainly by receptors for mannose 6-phosphate and galactose. (Evidence for this statement is that there is an intracellular increase in the β-glucuronidase level, for example in the liver).
As shown in Table 2, the sFv-huβ-Gluc contains relatively large amounts of galactose and, especially, mannose, which are mainly responsible for the binding to the particular receptors. The fusion protein/receptor complex which results and in which there is binding via the carbohydrate residues of the fusion protein is then removed from the extracellular compartment by internalization.
This rapid internalization mechanism, which is mainly mediated by galactose and mannose, is closely involved in the advantageous pharmacokinetics of the fusion protein according to the invention. These advantageous pharmacokinetics of the fusion protein to which galactose and, in particular, mannose are attached makes it possible for a hydrophilic prodrug which undergoes extracellular distribution to be administered i.v. at a relatively early time without eliciting non-specific prodrug activation. In this case an elimination step as described by Sharma et al. (Brit. J. Cancer, 61, 659, 1990) is unnecessary. Based on the data in Table 1, injection of a suitable prodrug (S. Adrianomenjanahari et al. 1992, Int. Carbohydrate Symp., Parts A264, 299) is possible even 3 days after injection of the sFv-huβ-Gluc fusion protein without producing significant side effects (data not shown).
A similarly advantageous attachment of carbohydrates to fusion proteins can also be achieved, for example, by secretory expression of the sFv-huβ-Gluc fusion protein in particular yeast strains such as Saccharomyces cerevisiae or Hansenula polymorpha. These organisms are capable of very effective mannosylation of fusion proteins which have appropriate N-glycosylation sites (Goochee et al., Biotechnology, 9, 1347-1354, 1991). Such fusion proteins which have undergone secretory expression in yeast cells show a high degree of mannosylation and favorable pharmacokinetics comparable to those of the sFv-huβ-Gluc fusion protein expressed in BHK cells (data not shown). In this case, the absence of galactose is compensated by the even higher degree of mannosylation of the fusion protein (Table 3). The sFv-huβ-Gluc fusion protein described above was constructed by genetic manipulation and expressed in yeast as described in detail in Example 9.
Instead of human β-glucuronidase it is, however, also possible to employ another glucuronidase with advantageous properties. For example, the E.coli β-glucuronidase has the particular advantage that its catalytic activity at pH 7.4 is significantly higher than that of human β-glucuronidase. In Example 10, an sFv-E.coli β-Gluc construct was prepared by methods of genetic manipulation and underwent secretory expression as functionally active mannosylated fusion protein in Saccharomyces cerevisiae. The pharmacokinetic data are comparable to those of the sFv-huβ-Gluc molecule which was expressed in yeast or in BHK cells (Table 1).
The glucuronidases from the fungus Kobayasia nipponica and from the plants Secale cereale have the advantage, for example, that they are also active as monomers. In Example 11, methods of genetic manipulation were used to prepare a construct which, after expression in Saccharomyces cerevisiae, excretes an sFv-B. cereus β-lactamase II fusion protein preferentially in mannosylated form.
This fusion protein likewise has, as the fusion proteins according to the invention, on the basis of β-glucuronidase pharmacokinetics which are favorable for prodrug activation (Table 1).
Furthermore, the compounds according to the invention can be employed not only in combination with a prodrug but also in the framework of conventional chemotherapy in which cytostatics which are metabolized as glucuronides and thus inactivated can be converted back into their toxic form by the administered compounds.
The following examples now describe the synthesis by genetic manipulation of sFv-β-Gluc fusion proteins, and the demonstration of the ability to function.
The starting material comprised the plasmids pABstop 431/26 hum VH and pABstop 431/26 hum VHL. These plasmids contain the humanized version of the VH gene and VL gene of anti-CEA MAb BW 431/26 (Güssow and Seemann, 1991, Meth. Enzymology, 203, 99-121). Further starting material comprised the plasmid pABstop 431/26 VH-huβ-Gluc 1H (EP-A2-0 501 215) which contains a VH exon, including the VH-intrinsic signal sequence, followed by a CHl exon, by the hinge exon of a human IgG3 C gene and the complete cDNA of human β-glucuronidase.